Pteridines are very important factors in human metabolism. Reduced pteridines are intermediates in anabolic and catabolic reactions and serve as coenzymes mediating the action of some enzymes. Pteridine and its derivatives play important roles in the synthesis of some vitamins, such as vitamin B2 and vitamin B10. Biopterin, neopterin, pterine, xanthopterin, and isoxanthopterin all belong to the pteridine family, which are well distributed in nature in living organisms.
Since pteridines are very important cofactors in the process of cell metabolism, their levels have significant importance in clinical diagnosis. They are excreted by humans in urine and the levels of pteridines have been found to elevate significantly when the cellular immune system is activated by certain diseases such as cancer, viral infections such as STDs and AIDS, and renal disease. Halpern, R. M. et al. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 587-591. Further investigation has shown that, for various tumors, quantitative alterations in the pteridine concentrations can be detected. Malignant tumors greatly disturb the biosynthesis and metabolism of pteridines, thus leading to a great change in pteridine concentrations. Each type of tumor shows its own pattern in changes of pteridine concentrations, since different pteridine derivatives may play various roles in different tumor-related disease. Goldberg, M. et al. Pteridines 1989, 1, 29-35.
Although the importance of various pteridines in cell metabolism of higher mammals was recognized decades ago, and some work had been carried out regarding the synthesis, reaction, and function of these compounds thereafter, the reason leading to the quantitative change of pteridines is still not fully understood. Kaufman, S. Proc. Natl. Acad. Sci. U.S.A. 1963, 50, 1085-1092. This is due primarily because the metabolism and regulation of pteridines have not been widely investigated due to the difficulties involved in quantitation.
High-performance liquid chromatography (HPLC) methods, such as cation-exchange chromatography, reversed-phase chromatography, and ion-pair chromatography have been employed as a tool for the quantitative measurement of pteridines. Woolf, J. H. et al. J. Chromatogr. 1983, 274, 398-402. However, these methods are time-consuming, not cost-effective, and have unsatisfactory separation. High-performance capillary electrophoresis (HPCE) is a very good alternative for HPLC due to its high efficiency, high speed, and small sample size requirements. HPCE coupled with UV spectrophotometric detection has been used as an alternative to separate different kinds of pteridines. Cha, K. W. et al. Pteridines 1993, 4, 210-213. However, this method is not suitable for practical purposes such as urine and serum analysis because the sensitivity of the UV detector is not high enough to detect the minute amount of pteridines in these kinds of clinical samples.
There is therefore a need in the art for a more accurate and sensitive method of detecting pteridines.
It is therefore a primary objective of the present invention to provide a method and means of detecting pteridines in clinical samples with sensitivity high enough to detect minute amounts of pteridines.
It is a further objective of the present invention to provide a method and means of detecting pteridines which is effective for use with clinical samples.
It is yet a further objective of the present invention to provide a method and means of detecting pteridines which is highly accurate.
It is still a further objective of the present invention to provide a method and means of detecting pteridines which has small sample size requirements.
It is a further objective of the present invention to provide a method and means of detecting pteridines which is fast and cost-effective.
These and other objectives will become apparent from the following detailed description of the invention.
The present invention sets forth a novel method and means for detecting pteridine levels in clinical samples. The method is based on high-performance capillary electrophoresis (CE) and laser-induced fluorescence (LIF) detection. CE provides better separation than high-performance liquid chromatography and the LIF detector enables the detection of minute amounts of pteridines in body fluid. The method is effective in detecting eight pteridine compounds at very low detection limits of less than 1xc3x9710xe2x88x9210 M, and as low as 1xc3x9710xe2x88x9211 M. At such detection limits, the method allows sufficient detection specificity and quantitation to diagnose elevated and decreased pteridine levels which are associated with cancer, and therefore may be used for purposes of cancer monitoring and for precancer screening.